Dynamic changes in the concentration of intracellular free calcium ([Ca2+])i, play an important role in both basal and secretagogue- regulated control of the prolactin (PRL) gene. In fact, the PRL gene is one of the first for which a primary effect of Ca2+ on gene transcription has been established. The PRL-secreting lactotrope, like other anterior pituitary cells, displays spontaneous oscillations of [Ca2+]i, which are driven by electrical activity and modulated by a variety of hypophysiotropic agents. However, the precise contribution of Ca2+ oscillations to the rate of PRL gene expression has remained obscure owing to the requirement for, and lack of, a strategy for dynamically monitoring both of these variables in the same, living cell. In this proposal, we plan to solve this problem by making "real-time" measurements of PRL gene expression in primary lactotropes that are also being subjected to [Ca2+]i, determinations by digital imaging, fluorescence microscopy of fura-2. Using this combined strategy, we aim to: 1) determine whether single cell shifts in oscillatory phenotypes are random or ordered events; 2) identify the signalling patterns (code) responsible for Ca2+ code of changes of gene expression. The potential health-related impact of these studies becomes obvious when one considers the ubiquity of [Ca2+]i as a signalling molecule along with its positioning at a point of convergence for other second messenger pathways. Given the analytical power at our disposal, the proposed experiments may well constitute the best chance yet to test directly the prevailing view that Ca2+-regulated processes are controlled by a code of [Ca2+]i oscillatory patterns which vary in frequency, amplitude, and/or shape.